The isolation and analysis of genes from samples of living organisms is an extremely effective means of performing research in the field of molecular biology. In the medical field as well, such analysis is widely used. In the past, numerous genes have been isolated and identified from living organisms and tissues for the purpose of genetic analysis. Furthermore, the polymerase chain reaction (PCR) method (Science: Vol. 239, pp. 487-491 (1988)) has been developed as a gene amplification method, and has made it possible to isolate and detect genes that are present in extremely small amounts. Furthermore, human genome analysis has developed rapidly, and associations between genetic mutations such as single-nucleotide polymorphism or the like and diseases or drug reactions have received attention. The utilization of such genetic polymorphism is not limited to molecular biology and genetics; it is expected that such polymorphism will be utilized in made-to-order therapy and drug administration in the field of medical diagnosis.
A typical gene analysis process comprises an extraction step and a detection step of the nucleic acid of interest from a sample; and optionally comprises a nucleic acid amplification step between the extraction step and the detection step.
A method that is widely known as a nucleic acid extraction method is the so-called protease K/phenol method (Molecular Cloning: A Laboratory Manual, Appendix E3-E4 (New York: Cold Spring Harbor Laboratory, 1989)), in which nucleic acids are liberated by destroying cells and breaking down proteins through the addition of a proteolytic enzyme such as protease K or the like, and a surfactant, after which phenol/chloroform is added, the nucleic acid is distributed into the aqueous phase by centrifugation, and ethanol, isopropanol or the like is added to the recovered aqueous phase so that the nucleic acid becomes insolubilized. However, this method is not suitable for automation, since it involves centrifugation step and the like. In order to solve such problems, methods that utilize the adsorption characteristics of nucleic acids on a solid phase have been proposed. For example, the bonding of nucleic acids to glass in the presence of chaotropic salts has been reported (Proc. Natl. Acad. Sci. USA 76, 615-619 (1979)). By applying such characteristics of nucleic acid bonding to silicon oxide (silica) under certain conditions, nucleic acid extraction kits and automated nucleic acid extraction devices based on magnetic silica particles, silica particles, silica fibers or filters, and spin columns, micro-plates or the like containing these substances, were disclosed (Japanese Patent Application Laid-Open (kohyo) No. H10-504834, Japanese Patent Application Laid-Open (kohyo) No. H9-505724, Japanese Patent Application Laid-Open (kokai) No. H8-320274, Japanese Patent Application Laid-Open (kokai) No. H9-304385, Japanese Patent Examined Publication (kokoku) No. 2807090).
In regard to methods used for nucleic acid detection step, and especially in regard to methods for detecting nucleic acids whose sequences differ by one or more nucleotides, single-stranded DNA conformational polymorphism (SSCP) or restriction enzyme fragment length polymorphism (RFLP) using gel electrophoresis, and microarray methods, DNA chip methods and bead array methods in which a single-stranded nucleic acid is fixed on glass, silicon wafers or various types of beads, and hybridization with the object nucleic acid is detected, as well as pyro-sequencing methods, invader methods, mass spectrometry methods, HPLC methods and the like have been developed and are widely utilized.
As nucleic acid amplification methods, polymerase chain reactions (PCR) (U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202), nucleic acid sequence-based amplification (NASBA) (European Patent NO. 0, 329, 822) and the like are known and utilized. Furthermore, nucleic acid determination methods using competitive amplification or co-amplification are generally known. In such methods, several types of internal control nucleic acids which react with the same primer as the nucleic acid of interest are subjected to an amplification reaction in the same vessel, and the amount of the nucleic acid of interest is determined using the known quantities of the internal control nucleic acids as a reference (Japanese Patent Application Laid-Open (kohyo) No. H8-107798, Japanese Patent Application Laid-Open (kohyo) No. H8-501222, Japanese Patent Application Laid-Open (kohyo) No. H11-123095, Japanese Patent Application Laid-Open (kohyo) No. H11-506613).
For the most part, such conventional techniques are utilized exclusively for the determination of the amount of the object nucleic acid of interest based on a process in which a fixed amount of a nucleic acid which can be detected while being discriminated from the nucleic acid of interest, and which is generally referred to as an internal control nucleic acid or the like, is amplified simultaneously with the nucleic acid of interest in the nucleic acid amplification step. In these methods, it is assumed throughout that the nucleic acid of interest has been prepared normally (Japanese Patent Application Laid-Open (kohyo) No. H8-107798, Japanese Patent Application Laid-Open (kohyo) No. H11-123095, Japanese Patent Application Laid-Open (kohyo) No. H11-506613). On the other hand, a method which reflects the extraction efficiency of the object nucleic acid in the extraction step, and a method for detecting cross-contamination between samples are described in Japanese Patent Application Laid-Open (kohyo) No. H8-501222. However, it is essential that the methods include a nucleic acid amplification step, and it is difficult to judge the success or failure of the overall series of nucleic acid analysis steps and/or to ascertain in which step an abnormality has occurred.
Accordingly, it is an object of the present invention to provide a method for analyzing a nucleic acid, whose aim is to ensure or improve the reliability of the analysis results obtained in the analysis of nucleic acids.